Modulation of L-selectin shedding via inhibition of tumor necrosis factor-α converting enzyme (TACE)

ABSTRACT

The present invention provides methods of modulating the shedding of L-selectin in cells or tissues using an inhibitor of TACE expression or activity. Antisense oligonucleotides targeted to nucleic acids encoding TACE are preferred forms of TACE inhibitors. These methods are believed to be useful both therapeutically and diagnostically and as research tools. The present invention further comprises methods of treating conditions associated with altered L-selectin shedding or altered L-selectin levels.

FIELD OF THE INVENTION

This invention is directed to modulation of TNF-αmediated cellularevents, especially shedding of L-selectin, by modulating the activity orexpression of TNF-α-converting enzyme (TACE). TACE is a disintegrinmetalloprotease involved in the processing of tumor necrosis factor α(TNF-α), a cytokine implicated in infectious and inflammatory disease.

BACKGROUND OF THE INVENTION

Tumor necrosis factor a (TNF-α, also cachectin) is an important cytokinethat plays a role in host defense. The cytokine is produced primarily inmacrophages and monocytes in response to infection, invasion, injury, orinflammation. Some examples of inducers of TNF-α include bacterialendotoxins, bacteria, viruses, lipopolysaccharide (LPS) and cytokinesincluding GM-CSF, IL-1, Il-2 and IFN-γ.

TNF-α is initially synthesized as a 26 kD membrane-bound protein. A 17kD fragment of TNF-α is secreted and forms a trimer with other secretedforms. This trimer interacts with two different receptors, TNF receptorI (TNFRI, p55) and TNFRII (p75), in order to transduce its effects—thenet result of which is altered gene expression and/or apoptosis.Cellular factors induced by TNF-α include interleukin-1 (IL-1),interleukin-6 (IL-6), interleukin-8 (IL-8), interferon-γ (IFN-γ),platelet derived growth factor (PDGF), epidermal growth factor (EGF),and endothelial cell adhesion molecules including endothelial leukocyteadhesion molecule 1 (ELAM-1), intercellular adhesion molecule-1 (ICAM-1)and vascular cell adhesion molecule-1 (VCAM-1) (Tracey, K. J., et al.,Annu. Rev. Cell Biol., 1993, 9, 317-343; Arvin, B., et al., Ann. NYAcad. Sci., 1995, 765, 62-71).

The processing of TNF-α from its membrane-bound form to its secretedform is due to a specific metalloprotease known as TNF-α convertingenzyme (TACE, also ADAM17). TACE is a member of the ADAM (A DisintegrinAnd Metalloprotease) family. TACE has also been shown to have a directproteolytic role in the processing of other membrane proteins includingp75 TNF receptor, L-selectin and transforming growth factor-α (TGF-α;Peschon, J. J., et al., Science, 1998, 282, 1281-1284). L-selectin, inparticular, is an adhesion molecule involved in leukocyte rolling andmediates the attachment of leukocytes to endothelium at sites ofinflammation as well as the binding of lymphocytes to high endothelialvenules of peripheral lymph nodes.

Inhibitors of TACE will inhibit release of mature TNF-α into theextracellular environment, preventing TNF-α mediated signaling. Thus,TACE inhibitors may have clinical utility in diseases associated withthe overproduction of TNF-α. Overexpression of TNF-α often results indisease states, particularly in infectious, inflammatory and autoimmunediseases. This process may involve the apoptotic pathways (Ksontini,R.,et al., J. Immunol., 1998, 160, 4082-4089). High levels of plasma TNF-αhave been found in infectious diseases such as sepsis syndrome,bacterial meningitis, cerebral malaria, and AIDS; autoimmune diseasessuch as rheumatoid arthritis, inflammatory bowel disease (includingCrohn's disease), sarcoidosis, multiple sclerosis, Kawasaki syndrome,graft-versus-host disease and transplant (allograft) rejection; organfailure conditions such as adult respiratory distress syndrome,congestive heart failure, acute liver failure and myocardial infarction(Eigler, A., et al., Immunol. Today, 1997, 18, 487-492). Other diseasesin which TNF-α is involved include asthma (Shah, A., et al., Clinicaland Experimental Allergy, 1995, 25, 1038-1044), brain injury followingischemia (Arvin, B., et al., Ann. NY Acad. Sci., 1995, 765, 62-71),non-insulin-dependent diabetes mellitus (Hotamisligil, G. S., et al.,Science, 1993, 259, 87-90), insulin-dependent diabetes mellitus (Yang,X.-D., et al., J. Exp. Med., 1994, 180, 995-1004), hepatitis (Ksontini,R., et al., J. Immunol., 1998, 160, 4082-4089), atopic dermatitis(Sumimoto, S., et al., Arch. Dis. Child., 1992, 67, 277-279), andpancreatitis (Norman, J. G., et al., Surgery, 1996, 120, 515-521).Further, Suganuma, M., et al. (Cancer Res., 1996, 56, 3711-3715) suggestthat inhibitors of TNF-α may be useful for cancer prevention. Inaddition, elevated TNF-α expression may play a role in obesity (Kern, P.A., J. Nutr., 1997, 127, 1917S-1922S). TNF-α was found to be expressedin human adipocytes and increased expression, in general, correlatedwith obesity.

L-selectin has been found to be involved in ischemia/reperfusion injury,especially myocardial (Ma, X. L., et al., Circulation, 1993, 88,649-658), and liver (Yadav, S. S., Am. J. Physiol., 1998, 275,G1341-G1352) and thromboembolic stroke (Bednar, M. M., et al., Neurol.Res., 1998, 20, 403-408); acute myeloid leukemia (Extermann, M., et al.,Blood, 1998, 92, 3115-3122), B-cell chronic lymphocytic leukemia(Csanaky, G., et al., Haematologica, 1994, 79, 132-136); experimentalautoimmune encephalomyelitis (EAE), an animal model of multiplesclerosis (Archelos, J. J., et al., J. Neurol. Sci., 1998, 159,127-134), human T-cell lymphotropic virus type I-associated myelopathy(Tsujino, A., et al., J. Neurol. Sci., 1998, 155, 76-79),meningoencephalitis (Buhrer, C., et al., Arch. Dis. Child., 1996, 74,288-292); rheumatoid arthritis (Kurohori, Y., et al., Clin. Rheumatol.,1995, 14, 335-341), ulcerative colitis (Seidelin, J. B., et al., Am. J.Gastroenterol., 1998, 93, 1854-1859); chronic lung disease (Kotecha, S.,et al., Arch. Dis. Child. Fetal Neonatal Ed., 1998, 78, F143-F147).

There are currently several approaches to inhibiting TNF-α expression.Approaches used to treat rheumatoid arthritis include a chimericanti-TNF-α antibody, a humanized monoclonal anti-TNF-α antibody, andrecombinant human soluble TNF-α receptor (Camussi, G., Drugs, 1998, 55,613-620). Other examples are indirect TNF-α inhibitors includingphosphodiesterase inhibitors (e.g. pentoxifylline) and metalloproteaseinhibitors (Eigler, A., et al., Immunol. Today, 1997, 18, 487-492). Anadditional class of a direct TNF-α inhibitor is oligonucleotides,including triplex-forming oligonucleotides, ribozymes, and antisenseoligonucleotides.

Inhibitors of L-selectin include monoclonal antibodies (Bednar, M. M.,et al., Neurol. Res., 1998, 20, 403-408), fucoidin (Nasu, T., et al.,Immunol. Lett., 1997, 59, 47-51), and oligonucleotide aptamers(Ringquist, S. and Parma, D., Cytometry, 1998, 33, 394-405).

A class of inducers of L-selectin shedding has been described which aredesigned to promote the clustering of L-selectin at the cell surface,resulting in the subsequent shedding of L-selectin. These compounds aremultivalent ligands dubbed “neoglycopolymers” which present multiplecopies of saccharide epitopes on an extended backbone. Gordon, E. J. etal., Nature, 1998, 392, 30-31.

L-selectin shedding can be inhibited by a hydroxamic acid-basedmetalloprotease inhibitor, KD-IX-73-4. Feehan et al., J. Biol. Chem.,1996, 271, 7019-7024. Metalloprotease inhibitors including hydroxamateshave also been shown to block TNFα processing and secretion. McGeehan etal., Nature, 1994, 370, 558-561; Gearing et al., Nature, 1994, 370,555-557; Mohler et al., Nature, 1994, 370, 218-220; Crowe et al., J.Exp. Med., 1995, 181, 1205-1210.

Although broad spectrum inhibitors of matrix metalloproteases areeffective in inhibiting TACE, specific inhibitors are desired forclinical use.

U.S. Pat. No. 5,629,285 describes small molecule inhibitors of TACEbased on peptidyl derivatives. WO 96/41624 describes the use ofantisense oligonucleotides to block expression of TACE, but nooligonucleotide sequences are disclosed.

There remains an unmet need for therapeutic compositions and methods forinhibition of TACE, and disease processes associated therewith.Particularly desired is modulation of L-selectin shedding throughinhibition of TACE.

SUMMARY OF THE INVENTION

The present invention provides methods of modulating the shedding ofL-selectin in cells or tissues using an inhibitor of TACE expression oractivity. These methods are believed to be useful both therapeuticallyand diagnostically and as tools, for example, for detecting anddetermining the role of L-selectin in various cell functions andphysiological processes and conditions and for diagnosing conditionsassociated with expression of L-selectin. The present invention furthercomprises methods of treating conditions associated with alteredL-selectin shedding or altered L-selectin levels.

DETAILED DESCRIPTION OF THE INVENTION

TNF-α plays an important regulatory role in the immune response tovarious foreign agents. Overexpression of TNF-α results in a number ofinfectious and inflammatory diseases. As such, this cytokine representsan attractive target for treatment of such diseases. In particular,modulation of the expression of TNF-α may be useful for the treatment ofdiseases such as Crohn's disease, diabetes mellitus, multiple sclerosis,rheumatoid arthritis, hepatitis, pancreatitis and asthma.

TACE is responsible for processing the membrane-bound form of TNF-α intoits secreted form. Thus, modulation of TACE is thought to an effectivemeans of modulating TNF-α processing and diseases or conditionsassociated with expression of TNF-α.

As disclosed herein, TACE is also responsible for the shedding ofL-selectin. Protein shedding is the proteolytic release of a cellsurface protein; it can serve a regulatory function by releasing solublemolecules into solution while decreasing their concentration on the cellsurface. Inhibition of TACE expression or activity is therefore alsobelieved to be an effective means of modulating L-selectin and diseasesor conditions associated with L-selectin. This includes diseases orconditions associated with L-selectin shedding per se but also includesdiseases associated with aberrant or undesired levels of L-selectin,particularly soluble L-selectin. For example, both leukocytes andendothelial cells have been found to shed L-selectin during acutemyocardial infarction. Soluble L-selectin is believed to be involved inthe immunological response to myocardial damage. Siminiak., J. et al.Exp. Clin. Cardiol. 1997, 2, 215-218.

As used herein, a “TACE inhibitor” or “TACE inhibitory compound” ismeant to include any compound which inhibits or decreases the activityor the expression (i.e., the synthesis) of TACE. Examples of suchcompounds include oligomeric compounds, particularly oligonucleoside oroligonucleotide compounds (including aptamers, ribozymes,triplex-forming oligonucleotides and antisense oligonucleotides), smallmolecule compounds (including peptidyl derivatives known in the art,such as hydroxamates or hydroxamic acid derivatives, including TAPI;thiols, phosphoryls and carboxyls), proteins, peptides or fragmentsthereof, and antibodies (including monoclonal antibodies).

In a preferred embodiment, the present invention employs antisenseoligonucleotides for use in modulating the function of nucleic acidmolecules encoding TACE, modulating the amount of TACE produced and,ultimately, the amount of L-selectin shedding. This is accomplished byproviding oligonucleotides which specifically hybridize with nucleicacids, preferably mRNA, encoding TACE.

This relationship between an antisense compound such as anoligonucleotide and its complementary nucleic acid target, to which ithybridizes, is commonly referred to as “antisense”. “Targeting” anoligonucleotide to a chosen nucleic acid target, in the context of thisinvention, is a multistep process. The process usually begins withidentifying a nucleic acid sequence whose function is to be modulated.This may be, as examples, a cellular gene (or mRNA made from the gene)whose expression is associated with a particular disease state, or aforeign nucleic acid from an infectious agent. In the present invention,the targets are nucleic acids encoding TACE; in other words, a geneencoding TACE, or mRNA expressed from the TACE gene. mRNA whichencodesTACE is presently the preferred target. The targeting processalso includes determination of a site or sites within the nucleic acidsequence for the antisense interaction to occur such that modulation ofgene expression will result.

In accordance with this invention, persons of ordinary skill in the artwill understand that messenger RNA includes not only the information toencode a protein using the three letter genetic code, but alsoassociated ribonucleotides which form a region known to such persons asthe 5′-untranslated region, the 3′-untranslated region, the 5′ capregion and intron/exon junction ribonucleotides. Thus, oligonucleotidesmay be formulated in accordance with this invention which are targetedwholly or in part to these associated ribonucleotides as well as to theinformational ribonucleotides. The oligonucleotide may therefore bespecifically hybridizable with a transcription initiation site region, atranslation initiation codon region, a 5′ cap region, an intron/exonjunction, coding sequences, a translation termination codon region orsequences in the 5′- or 3′-untranslated region. Since, as is known inthe art, the translation initiation codon is typically 5′-AUG (intranscribed mRNA molecules; 5′-ATG in the corresponding DNA molecule),the translation initiation codon is also referred to as the “AUG codon,”the “start codon” or the “AUG start codon.” A minority of genes have atranslation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function invivo. Thus, the terms “translation initiation codon” and “start codon”can encompass many codon sequences, even though the initiator amino acidin each instance is typically methionine (in eukaryotes) orformylmethionine (prokaryotes). It is also known in the art thateukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “start codon” and“translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA molecule transcribedfrom a gene encoding TACE, regardless of the sequence(s) of such codons.It is also known in the art that a translation termination codon (or“stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA,5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAGand 5′-TGA, respectively). The terms “start codon region,” “AUG region”and “translation initiation codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. This region is a preferred target region. Similarly,the terms “stop codon region” and “translation termination codon region”refer to a portion of such an MRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation termination codon. This region is a preferredtarget region. The open reading frame (ORF) or “coding region,” which isknown in the art to refer to the region between the translationinitiation codon and the translation termination codon, is also a regionwhich may be targeted effectively. Other preferred target regionsinclude the 5′ untranslated region (5′UTR), known in the art to refer tothe portion of an mRNA in the 5′ direction from the translationinitiation codon, and thus including nucleotides between the 5′ cap siteand the translation initiation codon of an mRNA or correspondingnucleotides on the gene and the 3′ untranslated region (3′UTR), known inthe art to refer to the portion of an mRNA in the 3′ direction from thetranslation termination codon, and thus including nucleotides betweenthe translation termination codon and 3′ end of an mRNA or correspondingnucleotides on the gene. The 5′ cap of an mRNA comprises anN7-methylated guanosine residue joined to the 5′-most residue of themRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA isconsidered to include the 5′ cap structure itself as well as the first50 nucleotides adjacent to the cap. The 5′ cap region may also be apreferred target region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma pre-mRNA transcript to yield one or more mature mRNA. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,exon-exon or intron-exon junctions, may also be preferred targetregions, and are particularly useful in situations where aberrantsplicing is implicated in disease, or where an overproduction of aparticular mRNA splice product is implicated in disease. Aberrant fusionjunctions due to rearrangements or deletions are also preferred targets.Targeting particular exons in alternatively spliced mRNAs may also bepreferred. It has also been found that introns can also be effective,and therefore preferred, target regions for antisense compoundstargeted, for example, to DNA or pre-mRNA.

Once the target site or sites have been identified, oligonucleotides arechosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity, to give thedesired modulation.

“Hybridization”, in the context of this invention, means hydrogenbonding, also known as Watson-Crick base pairing, between complementarybases, usually on opposite nucleic acid strands or two regions of anucleic acid strand. Guanine and cytosine are examples of complementarybases which are known to form three hydrogen bonds between them. Adenineand thymine are examples of complementary bases which form two hydrogenbonds between them.

“Specifically hybridizable” and “complementary” are terms which are usedto indicate a sufficient degree of complementarity such that stable andspecific binding occurs between the DNA or RNA target and theoligonucleotide.

It is understood that an oligonucleotide need not be 100% complementaryto its target nucleic acid sequence to be specifically hybridizable. Anoligonucleotide is specifically hybridizable when binding of theoligonucleotide to the target interferes with the normal function of thetarget molecule to cause a loss of utility, and there is a sufficientdegree of complementarity to avoid non-specific binding of theoligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment and, in the case of invitro assays, under conditions in which the assays are conducted.

Hybridization of antisense oligonucleotides with mRNA interferes withone or more of the normal functions of mRNA. The functions of mRNA to beinterfered with include all vital functions such as, for example,translocation of the RNA to the site of protein translation, translationof protein from the RNA, splicing of the RNA to yield one or more mRNAspecies, and catalytic activity which may be engaged in by the RNA.Binding of specific protein(s) to the RNA may also be interfered with byantisense oligonucleotide hybridization to the RNA.

The overall effect of interference with mRNA function is modulation ofexpression of TACE. In the context of this invention “modulation” meanseither inhibition or stimulation; i.e., either a decrease or increase inexpression. Inhibition is presently preferred. This inhibition can bemeasured in ways which are routine in the art, for example by Northernblot assay of mRNA expression, or reverse transcriptase PCR, as taughtin the examples of the instant application or by Western blot, ELISAassay or immunoprecipitation assay of protein expression, or flowcytometry as taught in the examples of the present application. Asdesired, effects on cell proliferation or tumor cell growth can also bemeasured. Measurement of L-selectin shedding can be measured by flowcytometry as taught in the examples hereinbelow.

Oligonucleotides can be used in diagnostics, therapeutics, prophylaxis,and as research reagents and in kits. Oligonucleotides which hybridizeto nucleic acids encoding TACE can be exploited in sandwich,calorimetric and other assays. Provision of means for detectinghybridization of oligonucleotide with the TACE gene or mRNA canroutinely be accomplished. Such provision may include enzymeconjugation, radiolabelling or any other suitable detection systems.Kits for detecting the presence or absence of TACE may also be prepared.

The present invention is also suitable for diagnosing abnormalinflammatory states in tissue or other samples from patients suspectedof having an inflammatory disease such as rheumatoid arthritis. Theability of the oligonucleotides of the present invention to inhibitinflammatory processes may be employed to diagnose such states. A numberof assays may be formulated, which assays will commonly comprisecontacting a tissue sample with an oligonucleotide under conditionsselected to permit detection and, usually, quantitation of suchinhibition. In the context of this invention, to “contact” tissues orcells with an oligonucleotide or oligonucleotides means to add theoligonucleotide(s), usually in a liquid carrier, to a cell suspension ortissue sample, either in vitro or ex vivo, or to administer theoligonucleotide(s) to cells or tissues within an animal.

The oligonucleotides of this invention may also be used for researchpurposes. Thus, the specific hybridization exhibited by theoligonucleotides may be used for assays, purifications, cellular productpreparations and in other methodologies which may be appreciated bypersons of ordinary skill in the art.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid or deoxyribonucleic acid.This term includes oligonucleotides composed of naturally-occurringnucleobases, sugars and covalent intersugar (backbone) linkages as wellas oligonucleotides having non-naturally-occurring portions whichfunction similarly. Such modified or substituted oligonucleotides areoften preferred over native forms because of desirable properties suchas, for example, enhanced cellular uptake, enhanced binding to targetand increased stability in the presence of nucleases.

The antisense compounds in accordance with this invention preferablycomprise from about 5 to about 50 nucleobases. Particularly preferredare antisense oligonucleotides comprising from about 8 to about 30nucleobases (i.e. from about 8 to about 30 linked nucleosides). As isknown in the art, a nucleoside is a base-sugar combination. The baseportion of the nucleoside is normally a heterocyclic base. The two mostcommon classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos.: 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.:5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNAcompounds can be found in Nielsen et al. (Science, 1991, 254,1497-1500).

Most preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N (CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O—, S—, or N-alkyl, O-alkyl-O-alkyl, O—, S—, orN-alkenyl, or O—, S— or N-alkynyl, wherein the alkyl, alkenyl andalkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)₂ON(CH₃)₂, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br,CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, poly-alkylamino, substituted silyl,an RNA cleaving group, a reporter groupf an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy (2′—O—CH₂CH₂OCH₃, alsoknown as 2′—O—(2-methoxyethyl) or 2′-MOE) (Martin et al. Helv. Chim.Acta 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferredmodification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂group, also known as 2′-DMAOE, and 2′-dimethylamino-ethoxyethoxy(2′-DMAEOE), i.e., 2′—O—CH₂—O—CH₂—N(CH₂)₂.

Other preferred modifications include 2′-methoxy (2′—O—CH₃),2′-aminopropoxy (2′—OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugars structures include, but are not limited to, U.S.Pat. Nos.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,0531 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C or m5c), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in the Concise Encyclopedia Of Polymer Science AndEngineering 1990, pages 858-859, Kroschwitz, J. I., ed. John Wiley &Sons, those disclosed by Englisch et al. (Angewandte Chemie,International Edition 1991, 30, 613-722), and those disclosed bySanghvi, Y. S., Chapter 15, Antisense Research and Applications 1993,pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press. Certain ofthese nucleobases are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications 1993, CRC Press, Boca Raton, pages 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205;5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; and 5,681,941.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. Such moieties include but are not limitedto lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA 1989, 86, 6553-6556), cholic acid (Manoharan etal., Bioorg. Med. Chem. Lett. 1994, 4, 1053-1059), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci. 1992, 660,306-309; Manoharan et al., Bioorg. Med. Chem. Let. 1993, 3, 2765-2770),a thiocholesterol (Oberhauser et al., Nucl. Acids Res. 1992, 20,533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J. 1991, 10, 1111-1118; Kabanov et al.,FEBS Lett. 1990, 259, 327-330; Svinarchuk et al., Biochimie 1993, 75,49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett. 1995, 36, 3651-3654; Shea et al.,Nucl. Acids Res. 1990, 18, 3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides& Nucleotides 1995, 14,969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett.1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta 1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther. 1996, 277, 923-937).

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

The present invention also includes oligonucleotides which are chimericoligonucleotides. “Chimeric” oligonucleotides or “chimeras,” in thecontext of this invention, are oligonucleotides which contain two ormore chemically distinct regions, each made up of at least onenucleotide. These oligonucleotides typically contain at least one regionwherein the oligonucleotide is modified so as to confer upon theoligonucleotide increased resistance to nuclease degradation, increasedcellular uptake, and/or increased binding affinity for the targetnucleic acid. An additional region of the oligonucleotide may serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNase H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof antisense inhibition of gene expression. Cleavage of the RNA targetcan be routinely detected by gel electrophoresis and, if necessary,associated nucleic acid hybridization techniques known in the art. ThisRNAse H-mediated cleavage of the RNA target is distinct from the use ofribozymes to cleave nucleic acids. Ribozymes are not comprehended by thepresent invention.

Examples of chimeric oligonucleotides include but are not limited to“gapmers,” in which three distinct regions are present, normally with acentral region flanked by two regions which are chemically equivalent toeach other but distinct from the gap. A preferred example of a gapmer isan oligonucleotide in which a central portion (the “gap”) of theoligonucleotide serves as a substrate for RNase H and is preferablycomposed of 2′-deoxynucleotides, while the flanking portions (the 5′ and3′ “wings”) are modified to have greater affinity for the target RNAmolecule but are unable to support nuclease activity (e.g., fluoro- or2′-O-methoxyethyl-substituted). Chimeric oligonucleotides are notlimited to those with modifications on the sugar, but may also includeoligonucleosides or oligonucleotides with modified backbones, e.g., withregions of phosphorothioate (P═S) and phosphodiester (P═O) backbonelinkages or with regions of MMI and P=S backbone linkages. Otherchimeras include “wingmers,” also known in the art as “hemimers,” thatis, oligonucleotides with two distinct regions. In a preferred exampleof a wingmer, the 5′ portion of the oligonucleotide serves as asubstrate for RNase H and is preferably composed of 2′-deoxynucleotides,whereas the 3′ portion is modified in such a fashion so as to havegreater affinity for the target RNA molecule but is unable to supportnuclease activity (e.g., 2′-fluoro- or 2′-O-methoxyethyl- substituted),or vice-versa. In one embodiment, the oligonucleotides of the presentinvention contain a 2′-O-methoxyethyl (2′—O—CH₂CH₂OCH₃) modification onthe sugar moiety of at least one nucleotide. This modification has beenshown to increase both affinity of the oligonucleotide for its targetand nuclease resistance of the oligonucleotide. According to theinvention, one, a plurality, or all of the nucleotide subunits of theoligonucleotides of the invention may bear a 2′-O-methoxyethyl(—O—CH₂CH₂OCH₃) modification. Oligonucleotides comprising a plurality ofnucleotide subunits having a 2′-O-methoxyethyl modification can havesuch a modification on any of the nucleotide subunits within theoligonucleotide, and may be chimeric oligonucleotides. Aside from or inaddition to 2′-O-methoxyethyl modifications, oligonucleotides containingother modifications which enhance antisense efficacy, potency or targetaffinity are also preferred. Chimeric oligonucleotides comprising one ormore such modifications are presently preferred.

The oligonucleotides used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including Applied Biosystems. Any other means for such synthesismay also be employed; the actual synthesis of the oligonucleotides iswell within the talents of the routineer. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and 2′-alkoxy or 2′-alkoxyalkoxy derivatives,including 2′-O-methoxyethyl oligonucleotides (Martin, P., Helv. Chim.Acta 1995, 78, 486-504). It is also well known to use similar techniquesand commercially available modified amidites and controlled-pore glass(CPG) products such as biotin, fluorescein, acridine orpsoralen-modified amidites and/or CPG (available from Glen Research,Sterling, Va.) to synthesize fluorescently labeled, biotinylated orother conjugated oligonucleotides.

The antisense compounds used in the present invention includebioequivalent compounds, including pharmaceutically acceptable salts andprodrugs. This is intended to encompass any pharmaceutically acceptablesalts, esters, or salts of such esters, or any other compound which,upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto pharmaceutically acceptable salts of the nucleic acids of theinvention and prodrugs of such nucleic acids. “Pharmaceuticallyacceptable salts” are physiologically and pharmaceutically acceptablesalts of the nucleic acids of the invention: i.e., salts that retain thedesired biological activity of the parent compound and do not impartundesired toxicological effects thereto (see, for example, Berge et al.,“Pharmaceutical Salts,” J. of Pharma Sci. 1977, 66, 1-19).

For oligonucleotides, examples of pharmaceutically acceptable saltsinclude but are not limited to (a) salts formed with cations such assodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

The oligonucleotides of the invention may additionally or alternativelybe prepared to be delivered in a “prodrug” form. The term “prodrug”indicates a therapeutic agent that is prepared in an inactive form thatis converted to an active form (i.e., drug) within the body or cellsthereof by the action of endogenous enzymes or other chemicals and/orconditions. In particular, prodrug versions of the oligonucleotides ofthe invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate]derivatives according to the methods disclosed in WO 93/24510 toGosselin et al., published Dec. 9, 1993.

For therapeutic or prophylactic treatment, oligonucleotides or otherTACE inhibitors are administered in accordance with this invention.Compounds may be formulated in a pharmaceutical composition, which mayinclude pharmaceutically acceptable carriers, thickeners, diluents,buffers, preservatives, surface active agents, neutral or cationiclipids, lipid complexes, liposomes, penetration enhancers, carriercompounds and other pharmaceutically acceptable carriers or excipientsand the like in addition to the oligonucleotide or other TACEinhibitor(s). Such compositions and formulations are comprehended by thepresent invention.

Pharmaceutical compositions comprising one or more TACE inhibitors mayinclude penetration enhancers in order to enhance the alimentarydelivery of the inhibitor(s). Penetration enhancers may be classified asbelonging to one of five broad categories, i.e., fatty acids, bilesalts, chelating agents, surfactants and non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems 1991, 8, 91-192;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems 1990, 7,1-33). One or more penetration enhancers from one or more of these broadcategories may be included.

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid,myristic acid, palmitic acid, stearic acid, linoleic acid, linolenicacid, dicaprate, tricaprate, recinleate, monoolein (a.k.a.1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, mono- and di-glycerides and physiologically acceptablesalts thereof (i.e., oleate, laurate, caprate, myristate, palmitate,stearate, linoleate, etc.) (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems 1990, 7, 1; El-Hariri et al., J. Pharm.Pharmacol. 1992 44, 651-654).

The physiological roles of bile include the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38In: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9thEd., Hardman et al., eds., McGraw-Hill, New York, N.Y., 1996, pages934-935). Various natural bile salts, and their synthetic derivatives,act as penetration enhancers. Thus, the term “bile salt” includes any ofthe naturally occurring components of bile as well as any of theirsynthetic derivatives.

Complex formulations comprising one or more penetration enhancers may beused. For example, bile salts may be used in combination with fattyacids to make complex formulations.

Chelating agents include, but are not limited to, disodiumethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines) [Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems 1990, 7, 1-33; Buur et al., J. ControlRel. 1990, 14, 43-51). Chelating agents have the added advantage of alsoserving as DNase inhibitors.

Surfactants include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems 1991, page92); and perfluorochemical emulsions, such as FC-43 (Takahashi et al.,J. Pharm. Phamacol. 1988, 40, 252-257).

Non-surfactants include, for example, unsaturated cyclic ureas, 1-alkyl-and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol. 1987, 39,621-626).

As used herein, “carrier compound” refers to a nucleic acid, or analogthereof, which is inert (i.e., does not possess biological activity perse) but is recognized as a nucleic acid by in vivo processes that reducethe bioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor.

In contrast to a carrier compound, a “pharmaceutically acceptablecarrier” (excipient) is a pharmaceutically acceptable solvent,suspending agent or any other pharmacologically inert vehicle fordelivering one or more oligonucleotide(s) or other TACE inhibitor(s) toan animal. The pharmaceutically acceptable carrier may be liquid orsolid and is selected with the planned manner of administration in mindso as to provide for the desired bulk, consistency, etc., when combinedwith a nucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutically acceptable carriers include, butare not limited to, binding agents (e.g., pregelatinized maize starch,polyvinyl-pyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrates (e.g., starch, sodiumstarch glycolate, etc.); or wetting agents (e.g., sodium laurylsulphate, etc.). Sustained release oral delivery systems and/or entericcoatings for orally administered dosage forms are described in U.S. Pat.Nos. 4,704,295; 4,556,552; 4,309,406; and 4,309,404.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional compatible pharmaceutically-activematerials such as, e.g., antipruritics, astringents, local anestheticsor anti-inflammatory agents, or may contain additional materials usefulin physically formulating various dosage forms of the composition ofpresent invention, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers. However,such materials, when added, should not unduly interfere with thebiological activities of the components of the compositions of theinvention.

Regardless of the method by which the TACE inhibitors are introducedinto a patient, colloidal dispersion systems may be used as deliveryvehicles to enhance the in vivo stability of the inhibitors and/or totarget the inhibitors to a particular organ, tissue or cell type.Colloidal dispersion systems include, but are not limited to,macromolecule complexes, nanocapsules, microspheres, beads andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, liposomes and lipid:oligonucleotide complexes ofuncharacterized structure. A preferred colloidal dispersion system foroligonucleotides is a plurality of liposomes. Liposomes are microscopicspheres having an aqueous core surrounded by one or more outer layersmade up of lipids arranged in a bilayer configuration (see, generally,Chonn et al., Current Op. Biotech. 1995, 6, 698-708).

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, epidermal, intradermal and transdermal), oral or parenteral.Parenteral administration includes intravenous drip, infusion orinjection, subcutaneous, intraperitoneal or intramuscular injection,pulmonary administration, e.g., by inhalation or insufflation, orintracranial, e.g., intrathecal or intraventricular, administration.Oligonucleotides with at least one 2′-O-methoxyethyl modification arebelieved to be particularly useful for oral administration.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable.

Compositions for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. In some cases it may be more effective to treat a patientwith an oligonucleotide of the invention in conjunction with othertraditional therapeutic modalities in order to increase the efficacy ofa treatment regimen. In the context of the invention, the term“treatment regimen” is meant to encompass therapeutic, palliative andprophylactic modalities. For example, a patient may be treated withconventional chemotherapeutic agents, particularly those used for tumorand cancer treatment. Such chemotherapeutic agents may be usedindividually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FUand oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide).

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in vitro andin in vivo animal models. In general, dosage is from 0.01 μg to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 μg to 100 g per kgof body weight, once or more daily, to once every 20 years.

Thus, in the context of this invention, by “therapeutically effectiveamount” is meant the amount of the compound which is required to have atherapeutic effect on the treated individual. This amount, which will beapparent to the skilled artisan, will depend upon the age and weight ofthe individual, the type of disease to be treated, perhaps even thegender of the individual, and other factors which are routinely takeninto consideration when designing a drug treatment. A therapeutic effectis assessed in the individual by measuring the effect of the compound onthe disease state in the animal. For example, if the disease to betreated is cancer, therapeutic effects are assessed by measuring therate of growth or the size of the tumor, or by measuring the productionof compounds such as cytokines, production of which is an indication ofthe progress or regression of the tumor.

The following examples illustrate the present invention and are notintended to limit the same.

EXAMPLES Example 1 Synthesis of Oligonucleotides

Unmodified oligodeoxynucleotides are synthesized on an automated DNAsynthesizer (Applied Biosystems model 380B) using standardphosphoramidite chemistry with oxidation by iodine.β-cyanoethyldiisopropyl-phosphoramidites are purchased from AppliedBiosystems (Foster City, Calif.). For phosphorothioate oligonucleotides,the standard oxidation bottle was replaced by a 0.2 M solution of³H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwisethiation of the phosphite linkages. The thiation cycle wait step wasincreased to 68 seconds and was followed by the capping step. Cytosinesmay be 5-methyl cytosines. (5-methyl deoxycytidine phosphoramiditesavailable from Glen Research, Sterling, Va. or Amersham PharmaciaBiotech, Piscataway, N.J.)

2′-methoxy oligonucleotides are synthesized using 2′-methoxyβ-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, Needham, Mass.) andthe standard cycle for unmodified oligonucleotides, except the wait stepafter pulse delivery of tetrazole and base is increased to 360 seconds.Other 2′-alkoxy oligonucleotides are synthesized by a modification ofthis method, using appropriate 2′-modified amidites such as thoseavailable from Glen Research, Inc., Sterling, Va.

2′-fluoro oligonucleotides are synthesized as described in Kawasaki etal. (J. Med. Chem. 1993, 36, 831-841). Briefly, the protected nucleosideN⁶-benzoyl-2′-deoxy-2′-fluoroadenosine is synthesized utilizingcommercially available 9-β-D-arabinofuranosyladenine as startingmaterial and by modifying literature procedures whereby the 2′-α-fluoroatom is introduced by a S_(N)2-displacement of a 2′-β-O-trifyl group.Thus N⁶-benzoyl-9-β-D-arabinofuranosyladenine is selectively protectedin moderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N⁶-benzoyl groups is accomplished usingstandard methodologies and standard methods are used to obtain the5′-dimethoxytrityl- (DMT) and 5′-DMT-3′-phosphoramidite intermediates.

The synthesis of 2′-deoxy-2′-fluoroguanosine is accomplished usingtetraisopropyldisiloxanyl (TPDS) protected 9-β-D-arabinofuranosylguanineas starting material, and conversion to the intermediatediisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS groupis followed by protection of the hydroxyl group with THP to givediisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation is followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies are used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

Synthesis of 2′-deoxy-2′-fluorouridine is accomplished by themodification of a known procedure in which 2,2′-anhydro-1-β-D-arabinofuranosyluracil is treated with 70% hydrogenfluoride-pyridine. Standard procedures are used to obtain the 5′-DMT and5′-DMT-3′phosphoramidites.

2′-deoxy-2′-fluorocytidine is synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN⁴-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures are used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

2′-(2-methoxyethyl)-modified amidites were synthesized according toMartin, P. (Helv. Chim. Acta 1995, 78, 486-506). For ease of synthesis,the last nucleotide may be a deoxynucleotide. 2′-O—CH₂CH₂OCH₃ cytosinesmay be 5-methyl cytosines.

Synthesis of 5-Methyl Cytosine Monomers

2,2′-Anhydro[1-(β-D-arabinofuranosyl)-5-methyluridine]:

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid which was crushed to a light tan powder (57 g, 85%crude yield). The material was used as is for further reactions.

2′-O-Methoxyethyl-5-methyluridine:

2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel was openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue was suspended in hot acetone (1 L). The insoluble saltswere filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) was dissolved in CH₃CN (600 mL) and evaporated. Asilica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3)containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 mL) andadsorbed onto silica (150 g) prior to loading onto the column. Theproduct was eluted with the packing solvent to give 160 g (63%) ofproduct.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine:

2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxy-trityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃CN (200mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/-Hexane/Acetone (5:5:1) containing 0.5% Et₃NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-uridine:

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by tlc by first quenching the tlc sample with the addition ofMeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine:

A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the later solution. The resulting reaction mixture wasstored overnight in a cold room. Salts were filtered from the reactionmixture and the solution was evaporated. The residue was dissolved inEtOAc (1 L) and the insoluble solids were removed by filtration. Thefiltrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturatedNaCl, dried over sodium sulfate and evaporated. The residue wastriturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine:

A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (tlc showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-cytidine:

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, tlc showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/Hexane (1:1) containing 0.5% Et₃NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite:

N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (tlc showed thereaction to be 95% complete) . The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAcHexane (3:1) as theeluting solvent. The pure fractions were combined to give 90.6 g (87%)of the title compound.

2′-O-(dimethylaminooxyethyl) Nucleoside Amidites

2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the artas 2′-O-(dimethylaminooxyethyl) nucleoside amidites] are prepared asdescribed in the following paragraphs. Adenosine, cytidine and guanosinenucleoside amidites are prepared similarly to the thymidine(5-methyluridine) except the exocyclic amines are protected with abenzoyl moiety in the case of adenosine and cytidine and with isobutyrylin the case of guanosine.

5′-O-tert-Butyldiphenylsilyl-O²2′-anhydro-5-methyluridine

O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g,0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) weredissolved in dry pyridine (500 ml) at ambient temperature under an argonatmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane(125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. Thereaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22,ethyl acetate) indicated a complete reaction. The solution wasconcentrated under reduced pressure to a thick oil. This was partitionedbetween dichloromethane (1 L) and saturated sodium bicarbonate (2×1L)and brine (1 L). The organic layer was dried over sodium sulfate andconcentrated under reduced pressure to a thick oil. The oil wasdissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) andthe solution was cooled to −10° C. The resulting crystalline product wascollected by filtration, washed with ethyl ether (3×200 mL) and dried(40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMRwere consistent with pure product.

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

In a 2 L stainless steel, unstirred pressure reactor was added borane intetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and withmanual stirring, ethylene glycol (350 mL, excess) was added cautiouslyat first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure<100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819, 86%).

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 hr the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eg.) was addedand the mixture for 1 hr. Solvent was removed under vacuum; residuechromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95, 78%).

5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine (1.77 g, 3.12 mmol) wasdissolved in a solution of 1 M pyridinium p-toluenesulfonate (PPTS) indry MeOH (30.6 mL). Sodium cyanoborohydride (0.39 g, 6.13 mmol) wasadded to this solution at 10° C. under inert atmosphere. The reactionmixture was stirred for 10 minutes at 10° C. After that the reactionvessel was removed from the ice bath and stirred at room temperature for2 hr, the reaction monitored by TLC (5% MeOH in CH₂Cl₂). Aqueous NaHCO₃solution (5%, 10 mL) was added and extracted with ethyl acetate (2×20mL). Ethyl acetate phase was dried over anhydrous Na₂SO₄, evaporated todryness. Residue was dissolved in a solution of 1 M PPTS in MeOH (30.6mL). Formaldehyde (20% w/w, 30 mL, 3.37 mmol) was added and the reactionmixture was stirred at room temperature for 10 minutes. Reaction mixturecooled to 10° C. in an ice bath, sodium cyanoborohydride (0.39 g, 6.13mmol) was added and reaction mixture stirred at 10° C. for 10 minutes.After 10 minutes, the reaction mixture was removed from the ice bath andstirred at room temperature for 2 hrs. To the reaction mixture 5% NaHCO₃(25 mL) solution was added and extracted with ethyl acetate (2×25 mL).Ethyl acetate layer was dried over anhydrous Na₂SO₄ and evaporated todryness . The residue obtained was purified by flash columnchromatography and eluted with 5% MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

2′-O-(dimethylaminooxyethyl)-5-methyluridine

Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dryTHF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). Thismixture of triethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) wasdried over P₂O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂O₅ under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

5-methyl-2′-deoxycytidine (5-me-C) containing oligonucleotides weresynthesized according to published methods (Sanghvi et al., Nucl. AcidsRes. 1993, 21, 3197-3203) using commercially available phosphoramidites(Glen Research, Sterling Va. or ChemGenes, Needham Mass.).

Oligonucleotides having methylene(methylimino) (MMI) backbones weresynthesized according to U.S. Pat. No. 5,378,825, which is coassigned tothe assignee of the present invention and is incorporated herein in itsentirety. For ease of synthesis, various nucleoside dimers containingMMI linkages were synthesized and incorporated into oligonucleotides.Other nitrogen-containing backbones are synthesized according to WO92/20823 which is also coassigned to the assignee of the presentinvention and incorporated herein in its entirety.

Oligonucleotides having amide backbones are synthesized according to DeMesmaeker et al. (Acc. Chem. Res. 1995, 28, 366-374). The amide moietyis readily accessible by simple and well-known synthetic methods and iscompatible with the conditions required for solid phase synthesis ofoligonucleotides.

Oligonucleotides with morpholino backbones are synthesized according toU.S. Pat. No. 5,034,506 (Summerton and Weller).

Peptide-nucleic acid (PNA) oligomers are synthesized according to P.E.Nielsen et al. (Science 1991, 254, 1497-1500).

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides are purified by precipitation twiceout of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotideswere analyzed by polyacrylamide gel electrophoresis on denaturing gelsand judged to be at least 85% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in synthesiswere periodically checked by ³¹P nuclear magnetic resonancespectroscopy, and for some studies oligonucleotides were purified byHPLC, as described by Chiang et al. (J. Biol. Chem. 1991, 266, 18162).Results obtained with HPLC-purified material were similar to thoseobtained with non-HPLC purified material.

Example 2 Human TACE Oligodeoxynucleotide Sequences

Antisense oligonucleotides were designed to target human TACE. Targetsequence data are from the TACE cDNA sequence published by Black, R. A.et al. (Nature, 1997, 385, 729-733); Genbank accession number U69611,provided herein as SEQ ID NO: 1. Oligodeoxynucleotides were synthesizedas uniformly phosphorothioate chimeric oligonucleotides having regionsof five 2′-O-methoxyethyl (2′-MOE) nucleotides at the wings and acentral region of ten deoxynucleotides. Oligonucleotide sequences areshown in Table 1. Oligonucleotide 14834 (SEQ ID NO. 14) is an antisenseoligodeoxynucleotide targeted to the human tumor necrosis factor-α(TNF-α) and was used as a positive control.

The human Jurkat T cell line and the human promonocytic leukaemia cellline, THP-1 (American Type Culture Collection, Manassas, Va.) weremaintained in RPMI 1640 growth media supplemented with 10% fetal calfserum (FCS; Life Technologies, Rockville, Md.). HUVEC cells (Clonetics,San Diego, Calif.) were cultivated in endothelial basal mediasupplemented with 10% fetal bovine serum (Hyclone, Logan, Utah).

NeoHK cells, human neonatal foreskin keratinocytes (obtained fromCascade Biologicals, Inc., Portland, Oreg.) were cultured inKeratinocyte Serum Free (SFM) medium containing the human recombinantEpidermal Growth Factor 1-53 and Bovine Pituitary Extract (LifeTechnologies, Rockville, Md.). For NeoHK cells, the cells were usedbetween passages 2 to 6.

HUVEC and NeoHK cells were allowed to reach 75% confluency prior to use.The cells were washed twice with warm (37° C.) OPTI-MEM™ (LifeTechnologies) . The cells were incubated in the presence of theappropriate culture medium, without the growth factors added, and theoligonucleotide formulated in LIPOFECTIN® (Life Technologies), a 1:1(w/w) liposome formulation of the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA),and dioleoyl phosphotidylethanolamine (DOPE) in membrane filtered water.For an initial screen, the oligonucleotide concentration was 300 nM in10 μg/ml LIPOFECTIN®. Treatment was for four hours. After treatment, themedium was removed and the cells were further incubated in culturemedium containing growth factors and 100 nM phorbol 12-myristate13-acetate (PMA, Sigma, St. Louis, Mo.). mRNA was analyzed 4 hourspost-induction with PMA. HUVEC cells were treated with 100 nMoligonucleotide in 10 μg/ml LIPOFECTIN®. HUVEC cells were not induced,but allowed to rest in normal growth media for 4 hours.

Jurkat and THP-1 cells were grown to approximately 75% confluency andresuspended in culture media at a density of 1×10⁷ cells/ml. A total of3.6×10⁶ cells were employed for each treatment by combining 360 μl ofcell suspension with oligonucleotide at the indicated concentrations toreach a final volume of 400 μl. Cells were then transferred to anelectroporation cuvette and electroporated using an ElectrocellManipulator 600 instrument (Biotechnologies and Experimental Research,Inc.) employing 350 V, 100 μF, at 13 Ω. Electroporated cells were thentransferred to conical tubes containing 5 ml of culture media, mixed byinversion, and plated onto 10 cm culture dishes.

Total mRNA was isolated using the RNEASY® Mini Kit (Qiagen, Valencia,Calif.; similar kits from other manufacturers may also be used),separated on a 1% agarose gel, transferred to HYBOND™-N+ membrane(Amersham Pharmacia Biotech, Piscataway, N.J.), a positively chargednylon membrane, and probed. A TACE probe was made using PCRamplification with the following primers: TS-17465′-GTGTCCTACTGCACAGGTAATAGC-3′ SEQ ID NO. 15 BS-24895′-AATGACTTGGCAGCTGTGCTGCT-3′ SEQ ID NO. 16 A glyceraldehyde 3-phosphatedehydrogenase (G3PDH) probe was purchased from Clontech (Palo Alto,Calif.), Catalog Number 9805-1. The fragments were purified fromlow-melting temperature agarose, as described in Maniatis, T., et al.,Molecular Cloning: A Laboratory Manual, 1989 and labeled with REDIVUE™³²P-dCTP (Amersham Pharmacia Biotech, Piscataway, N.J.) and Strip-EZlabelling kit (Ambion, Austin, Tex.). mRNA was quantitated by aPhosphoImager (Molecular Dynamics, Sunnyvale, Calif.).

Protein levels were measured using flow cytometry analysis. Followingoligonucleotide treatment, cells were detached from the plates andanalyzed for surface expression of cell adhesion molecules using aBecton Dickinson (San Jose, Calif.) FACScan. TACE anti-serum wasprepared as described below. Human L-selectin (CD62L) monoclonalantibody and goat anti-mouse antibody were obtained from BectonDickinson. TNF-α antibody was obtained from R & D Systems (Minneapolis,Minn.). Cell surface expression was calculated using the mean value offluorescence intensity using 3,000-5,000 cells stained with theappropriate antibody for each sample and time point. Results areexpressed as percentage of control (cell surface expression in cellsthat were not treated with oligonucleotides) based upon meanfluorescence intensity.

Preparation of TACE Anti-serum

An 11 mer peptide (RADPDPMKNTC) (SEQ ID NO: 17) corresponding to theN-terminus of the human Tumor Necrosis Factor-Alpha Converting Enzyme(TACE) was synthesized. The peptide was reduced with the REDUCE-IMM™Reducing kit (Pierce Chemical Company, rockford, IL) and coupled toIMJECT® maleimide-activated KLH (Pierce Chemical Company). This solutionwas allowed to react for two hours at room temperature and then dialyzedversus PBS.

Female NZW rabbits were chosen for production of polyclonal anti-serum.Preimmune serum was obtained just prior to immunization withTACEpep-KLH. Immunization was as follows: 500 μg TACEpep-KLH emulsifiedwith an equal volume of TITERMAX™ adjuvant (Sigma Chemical Company, St.Louis, Mo.) was injected intradermally into approximately 10 sites alongthe rabbits back. The animals were boosted at weeks 3, 5 and 11 with anequal volume emulsion. Booster shots at weeks 3 and 5 contained 500 mgpeptide and the boost at week 5 contained 250 mg. Serum samples wereobtained at weeks 8, 12 and 15, with the week 15 bleed being a terminalbleed.

TABLE 1 Nucleotide Sequences of Chimeric (deoxy gapped) 2′-O-methoxyethyl Human TACE Antisense Oligonucleotides SEQ TARGET GENE GENEISIS NUCLEOTIDE SEQUENCE¹ ID NUCLEOTIDE TARGET NO. (5′ −> 3′) NO:CO-ORDINATES² REGION 16337 CCTAGTCAGTGCTGTTATCA  2 2911-2930 3′-UTR16338 GCTGTGATTGATTGTAGGTC  3 2641-2660 3′-UTR 16339AGCATCTGCTAAGTCACTTC  4 2681-2700 3′-UTR 16340 AGCTGAGAACTAAATTAGCA  52584-2603 stop 16341 TGAGAACTAAATTAGCACTC  6 2581-2600 stop 16342TTAGCACTCTGTTTCTTTGC  7 2570-2589 stop 16343 CTGCAGTTTAAAGGAGGCAG  82531-2550 coding 16344 CTGTCAACACGATTCTGACG  9 2551-2570 coding 16345AAATGACTTGGCAGCTGTGC 10 2471-2490 coding 16346 AACCACGCTGGTCAGGAATA 110131-0150 coding 16347 ATAGGAGAGACTGCCTCATG 12 0114-0133 AUG 17965CCTAGTATGTGCTGCTATCA 13 mismatch control 14834 GGATGTTCGTCCTCCTCACA 14TNF-α control ¹Emboldened residues are 2′-methoxyethoxy residues (othersare 2′-deoxy-). All 2′-methoxyethoxy cytidines are 5-methyl-cytidines;all linkages are phosphorothioate linkages. ²Co-ordinates from GenbankAccession No. U69611, locus name “HSU69611”, SEQ ID NO. 1.

Result for an initial screen in HUVEC cells are shown in Table 2. Alloligonucleotides tested inhibited TACE mRNA expression greater than 70%.

TABLE 2 Inhibition of Human TACE mRNA Expression in HUVEC by Chimeric(2′-deoxy) Gapped Oligonucleotides SEQ GENE ISIS ID TARGET % mRNA % mRNANo: NO: REGION EXPRESSION INHIBITION basal — — 100.0%  — 16337 2 3′-UTR13.8% 86.2% 16338 3 3′-UTR 27.5% 72.5% 16339 4 3′-UTR 22.1% 77.9% 163405 stop 18.2% 81.8% 16341 6 stop 11.8% 88.2% 16342 7 stop 15.9% 84.1%16343 8 coding 20.1% 79.9% 16344 9 coding 13.0% 87.0% 16345 10  coding10.4% 89.6% 16346 11  coding 18.4% 81.6% 16347 12  AUG 23.7% 76.3%

Result for an initial screen in NeoHK cells are shown in Table 3. Alloligonucleotides tested inhibited TACE mRNA expression by greater than55%.

TABLE 3 Inhibition of Human TACE mRNA Expression in NeoHK Cells byChimeric (2′-deoxy) Gapped Oligonucleotides SEQ GENE ISIS ID TARGET %mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION basal — — 100.0%  —induced — — 104.0%  — 16337 2 3′-UTR 18.3% 81.7% 16338 3 3′-UTR 41.2%58.8% 16339 4 3′-UTR 23.5% 76.5% 16340 5 stop 29.8% 70.2% 16341 6 stop22.5% 77.5% 16342 7 stop 20.6% 79.4% 16343 8 coding 35.8% 64.2% 16344 9coding 15.3% 84.7% 16345 10  coding 25.2% 74.8% 16346 11  coding 40.6%59.4% 16347 12  AUG 21.4% 78.6%

Example 3 Dose Response of Chimeric (2′-deoxy) Gapped AntisensePhosphorothioate Oligonucleotide Effects on Human TACE mRNA Levels

ISIS 6337 (SEQ ID NO. 2) was chosen for further studies including doseresponse assays. For dose response, cells were treated as described inExample 2, except the concentration of oligonucleotide was varied.LIPOFECTIN® was added at a 10 μg/ml. The control included LIPOFECTIN® ata concentration of 10 μg/ml. In HUVEC cells, the IC50 was approximately50 nM, while in neoHK cells, the IC50 was 150 nM. Controloligonucleotides, targeting human TNF-α or mouse CD18, had no effect onTACE mRNA levels.

Example 4 Time Course of Chimeric (2′-deoxy) Gapped AntisensePhosphorothioate Oligonucleotide Effects on Human TACE mRNA Levels

To investigate the role of TACE in cell processes, the effect of TACEoligonucleotides on mRNA and protein levels over time was determined.Jurkat and THP-1 cells were treated with 20 μM oligonucleotide asdescribed in Example 2. Results are shown in Table 4.

TABLE 4 Inhibition of Human TACE mRNA and protein Expression in JurkatCells by Chimeric (2′-deoxy) Gapped Oligonucleotides SEQ GENE ISIS IDTARGET TIME % mRNA % protein No: NO: REGION (hours) EXPRESSIONEXPRESSION basal — — — 100.0% 100.0% 16337 2 3′-UTR 24  53.5%  22.3% ″ ″″ 48  30.1%  29.0% ″ ″ ″ 72  41.7%  36.9% 17965 13  control 24 104.4% 95.9% ″ ″ ″ 48 104.8% 101.2% ″ ″ ″ 72  95.8%  92.3%

ISIS 16337 (SEQ ID NO. 2) reduced both TACE mRNA and protein levels at24, 48, and 72 hours after transfection in Jurkat cells. Treatment withthe TACE antisense oligonucleotide resulted in greater than 75%inhibition of protein expression, which remained suppressed for up to 72hours. TACE mRNA levels were reduced by about 70% at 48 hours andremained suppressed at 72 hours. The 3 base mismatch contrololignucleotide, ISIS 17965 (SEQ ID NO. 13), had no effect on either TACEmRNA or protein levels. Similar results were seen in THP-1 cells.

Examples 5 Effect of TACE Antisense Oligonucleotides on L-selectinShedding

The role of TACE in promoting PMA-induced L-selectin shedding wasinveatigated in Jurkat cells. Jurkat cells were electroporated with 20μM of igonucleotide as described in Example 2. 6.4×10⁵ cells wereelectroporated in OPTI-MEM™ I media (Life Technologies) containing 1%FCS (Life Technologies) at room temperature with an electric fieldstrength of 750 V/cm. 24 hours after oligonucleotide treatment,L-selectin shedding was induced with 100 nM PMA (Calbiochem, ) for 5minutes at 37° C. L-selectin cell surface expression was analyzed byflow cytometry using a FACScan as described in Example 2.

Result are shown in Table 5.

TABLE 5 Inhibition of L-selectin Shedding in Jurkat Cells by Chimeric(2′-deoxy) Gapped TACE Antisense Oligonucleotides % L- % L- ISIS SEQ IDGENE TARGET TIME selectin selectin No: NO: REGION (hours) expressionexpression basal — — — 100.0% — E.P.¹ — — 24 18.3% 81.7% ″ ″ ″ 48 33.6%66.4% ″ ″ ″ 72 31.0% 69.0% 16337 2 3′-UTR 24 95.6%  4.4% ″ ″ ″ 48 96.0% 4.0% ″ ″ ″ 72 97.4%  2.6% 17965 13  control 24 17.1% 82.9% ″ ″ ″ 4832.2% 67.8% ″ ″ ″ 72 35.9% 64.1% ¹E.P. refers to cells electroporatedwithout oligonucleotide

In cells in which TACE expression had been reduced by the TACE antisenseoligonucleotide, treatment with PMA resulted in a dramatic decrease inL-selectin shedding. In contrast, in cells that were treated with thecontrol or the TNF-α antisense oligonucleotide, PMA still inducedL-selectin shedding. ISIS 16337 (SEQ ID NO. 2) did not alter the surfaceexpression of other adhesion molecules including CD3, CD45, LFA-1 or α4integrin, either in the presence or absence of PMA induction.

17 1 3014 DNA Homo sapiens CDS (115)..(2589) Nature 385 6618 729-7331997-02-20 U69611/Genbank 1997-04-09 1 ggattgaggg gctaggccgg gcggatcccgtcctcccccg atgtgagcag ttttccgaaa 60 ccccgtcagg cgaaggctgc ccagagaggtggagtcggta gcggggccgg gaac atg 117 Met 1 agg cag tct ctc cta ttc ctg accagc gtg gtt cct ttc gtg ctg gcg 165 Arg Gln Ser Leu Leu Phe Leu Thr SerVal Val Pro Phe Val Leu Ala 5 10 15 ccg cga cct ccg gat gac ccg ggc ttcggc ccc cac cag aga ctc gag 213 Pro Arg Pro Pro Asp Asp Pro Gly Phe GlyPro His Gln Arg Leu Glu 20 25 30 aag ctt gat tct ttg ctc tca gac tac gatatt ctc tct tta tct aat 261 Lys Leu Asp Ser Leu Leu Ser Asp Tyr Asp IleLeu Ser Leu Ser Asn 35 40 45 atc cag cag cat tcg gta aga aaa aga gat ctacag act tca aca cat 309 Ile Gln Gln His Ser Val Arg Lys Arg Asp Leu GlnThr Ser Thr His 50 55 60 65 gta gaa aca cta cta act ttt tca gct ttg aaaagg cat ttt aaa tta 357 Val Glu Thr Leu Leu Thr Phe Ser Ala Leu Lys ArgHis Phe Lys Leu 70 75 80 tac ctg aca tca agt act gaa cgt ttt tca caa aatttc aag gtc gtg 405 Tyr Leu Thr Ser Ser Thr Glu Arg Phe Ser Gln Asn PheLys Val Val 85 90 95 gtg gtg gat ggt aaa aac gaa agc gag tac act gta aaatgg cag gac 453 Val Val Asp Gly Lys Asn Glu Ser Glu Tyr Thr Val Lys TrpGln Asp 100 105 110 ttc ttc act gga cac gtg gtt ggt gag cct gac tct agggtt cta gcc 501 Phe Phe Thr Gly His Val Val Gly Glu Pro Asp Ser Arg ValLeu Ala 115 120 125 cac ata aga gat gat gat gtt ata atc aga atc aac acagat ggg gcc 549 His Ile Arg Asp Asp Asp Val Ile Ile Arg Ile Asn Thr AspGly Ala 130 135 140 145 gaa tat aac ata gag cca ctt tgg aga ttt gtt aatgat acc aaa gac 597 Glu Tyr Asn Ile Glu Pro Leu Trp Arg Phe Val Asn AspThr Lys Asp 150 155 160 aaa aga atg tta gtt tat aaa tct gaa gat atc aagaat gtt tca cgt 645 Lys Arg Met Leu Val Tyr Lys Ser Glu Asp Ile Lys AsnVal Ser Arg 165 170 175 ttg cag tct cca aaa gtg tgt ggt tat tta aaa gtggat aat gaa gag 693 Leu Gln Ser Pro Lys Val Cys Gly Tyr Leu Lys Val AspAsn Glu Glu 180 185 190 ttg ctc cca aaa ggg tta gta gac aga gaa cca cctgaa gag ctt gtt 741 Leu Leu Pro Lys Gly Leu Val Asp Arg Glu Pro Pro GluGlu Leu Val 195 200 205 cat cga gtg aaa aga aga gct gac cca gat ccc atgaag aac acg tgt 789 His Arg Val Lys Arg Arg Ala Asp Pro Asp Pro Met LysAsn Thr Cys 210 215 220 225 aaa tta ttg gtg gta gca gat cat cgc ttc tacaga tac atg ggc aga 837 Lys Leu Leu Val Val Ala Asp His Arg Phe Tyr ArgTyr Met Gly Arg 230 235 240 ggg gaa gag agt aca act aca aat tac tta atagag cta att gac aga 885 Gly Glu Glu Ser Thr Thr Thr Asn Tyr Leu Ile GluLeu Ile Asp Arg 245 250 255 gtt gat gac atc tat cgg aac act tca tgg gataat gca ggt ttt aaa 933 Val Asp Asp Ile Tyr Arg Asn Thr Ser Trp Asp AsnAla Gly Phe Lys 260 265 270 ggc tat gga ata cag ata gag cag att cgc attctc aag tct cca caa 981 Gly Tyr Gly Ile Gln Ile Glu Gln Ile Arg Ile LeuLys Ser Pro Gln 275 280 285 gag gta aaa cct ggt gaa aag cac tac aac atggca aaa agt tac cca 1029 Glu Val Lys Pro Gly Glu Lys His Tyr Asn Met AlaLys Ser Tyr Pro 290 295 300 305 aat gaa gaa aag gat gct tgg gat gtg aagatg ttg cta gag caa ttt 1077 Asn Glu Glu Lys Asp Ala Trp Asp Val Lys MetLeu Leu Glu Gln Phe 310 315 320 agc ttt gat ata gct gag gaa gca tct aaagtt tgc ttg gca cac ctt 1125 Ser Phe Asp Ile Ala Glu Glu Ala Ser Lys ValCys Leu Ala His Leu 325 330 335 ttc aca tac caa gat ttt gat atg gga actctt gga tta gct tat gtt 1173 Phe Thr Tyr Gln Asp Phe Asp Met Gly Thr LeuGly Leu Ala Tyr Val 340 345 350 ggc tct ccc aga gca aac agc cat gga ggtgtt tgt cca aag gct tat 1221 Gly Ser Pro Arg Ala Asn Ser His Gly Gly ValCys Pro Lys Ala Tyr 355 360 365 tat agc cca gtt ggg aag aaa aat atc tatttg aat agt ggt ttg acg 1269 Tyr Ser Pro Val Gly Lys Lys Asn Ile Tyr LeuAsn Ser Gly Leu Thr 370 375 380 385 agc aca aag aat tat ggt aaa acc atcctt aca aag gaa gct gac ctg 1317 Ser Thr Lys Asn Tyr Gly Lys Thr Ile LeuThr Lys Glu Ala Asp Leu 390 395 400 gtt aca act cat gaa ttg gga cat aatttt gga gca gaa cat gat ccg 1365 Val Thr Thr His Glu Leu Gly His Asn PheGly Ala Glu His Asp Pro 405 410 415 gat ggt cta gca gaa tgt gcc ccg aatgag gac cag gga ggg aaa tat 1413 Asp Gly Leu Ala Glu Cys Ala Pro Asn GluAsp Gln Gly Gly Lys Tyr 420 425 430 gtc atg tat ccc ata gct gtg agt ggcgat cac gag aac aat aag atg 1461 Val Met Tyr Pro Ile Ala Val Ser Gly AspHis Glu Asn Asn Lys Met 435 440 445 ttt tca aac tgc agt aaa caa tca atctat aag acc att gaa agt aag 1509 Phe Ser Asn Cys Ser Lys Gln Ser Ile TyrLys Thr Ile Glu Ser Lys 450 455 460 465 gcc cag gag tgt ttt caa gaa cgcagc aat aaa gtt tgt ggg aac tcg 1557 Ala Gln Glu Cys Phe Gln Glu Arg SerAsn Lys Val Cys Gly Asn Ser 470 475 480 agg gtg gat gaa gga gaa gag tgtgat cct ggc atc atg tat ctg aac 1605 Arg Val Asp Glu Gly Glu Glu Cys AspPro Gly Ile Met Tyr Leu Asn 485 490 495 aac gac acc tgc tgc aac agc gactgc acg ttg aag gaa ggt gtc cag 1653 Asn Asp Thr Cys Cys Asn Ser Asp CysThr Leu Lys Glu Gly Val Gln 500 505 510 tgc agt gac agg aac agt cct tgctgt aaa aac tgt cag ttt gag act 1701 Cys Ser Asp Arg Asn Ser Pro Cys CysLys Asn Cys Gln Phe Glu Thr 515 520 525 gcc cag aag aag tgc cag gag gcgatt aat gct act tgc aaa ggc gtg 1749 Ala Gln Lys Lys Cys Gln Glu Ala IleAsn Ala Thr Cys Lys Gly Val 530 535 540 545 tcc tac tgc aca ggt aat agcagt gag tgc ccg cct cca gga aat gct 1797 Ser Tyr Cys Thr Gly Asn Ser SerGlu Cys Pro Pro Pro Gly Asn Ala 550 555 560 gaa gat gac act gtt tgc ttggat ctt ggc aag tgt aag gat ggg aaa 1845 Glu Asp Asp Thr Val Cys Leu AspLeu Gly Lys Cys Lys Asp Gly Lys 565 570 575 tgc atc cct ttc tgc gag agggaa cag cag ctg gag tcc tgt gca tgt 1893 Cys Ile Pro Phe Cys Glu Arg GluGln Gln Leu Glu Ser Cys Ala Cys 580 585 590 aat gaa act gac aac tcc tgcaag gtg tgc tgc agg gac ctt tcc ggc 1941 Asn Glu Thr Asp Asn Ser Cys LysVal Cys Cys Arg Asp Leu Ser Gly 595 600 605 cgc tgt gtg ccc tat gtc gatgct gaa caa aag aac tta ttt ttg agg 1989 Arg Cys Val Pro Tyr Val Asp AlaGlu Gln Lys Asn Leu Phe Leu Arg 610 615 620 625 aaa gga aag ccc tgt acagta gga ttt tgt gac atg aat ggc aaa tgt 2037 Lys Gly Lys Pro Cys Thr ValGly Phe Cys Asp Met Asn Gly Lys Cys 630 635 640 gag aaa cga gta cag gatgta att gaa cga ttt tgg gat ttc att gac 2085 Glu Lys Arg Val Gln Asp ValIle Glu Arg Phe Trp Asp Phe Ile Asp 645 650 655 cag ctg agc atc aat actttt gga aag ttt tta gca gac aac atc gtt 2133 Gln Leu Ser Ile Asn Thr PheGly Lys Phe Leu Ala Asp Asn Ile Val 660 665 670 ggg tct gtc ctg gtt ttctcc ttg ata ttt tgg att cct ttc agc att 2181 Gly Ser Val Leu Val Phe SerLeu Ile Phe Trp Ile Pro Phe Ser Ile 675 680 685 ctt gtc cat tgt gtg gataag aaa ttg gat aaa cag tat gaa tct ctg 2229 Leu Val His Cys Val Asp LysLys Leu Asp Lys Gln Tyr Glu Ser Leu 690 695 700 705 tct ctg ttt cac cccagt aac gtc gaa atg ctg agc agc atg gat tct 2277 Ser Leu Phe His Pro SerAsn Val Glu Met Leu Ser Ser Met Asp Ser 710 715 720 gca tcg gtt cgc attatc aaa ccc ttt cct gcg ccc cag act cca ggc 2325 Ala Ser Val Arg Ile IleLys Pro Phe Pro Ala Pro Gln Thr Pro Gly 725 730 735 cgc ctg cag cct gcccct gtg atc cct tcg gcg cca gca gct cca aaa 2373 Arg Leu Gln Pro Ala ProVal Ile Pro Ser Ala Pro Ala Ala Pro Lys 740 745 750 ctg gac cac cag agaatg gac acc atc cag gaa gac ccc agc aca gac 2421 Leu Asp His Gln Arg MetAsp Thr Ile Gln Glu Asp Pro Ser Thr Asp 755 760 765 tca cat atg gac gaggat ggg ttt gag aag gac ccc ttc cca aat agc 2469 Ser His Met Asp Glu AspGly Phe Glu Lys Asp Pro Phe Pro Asn Ser 770 775 780 785 agc aca gct gccaag tca ttt gag gat ctc acg gac cat ccg gtc acc 2517 Ser Thr Ala Ala LysSer Phe Glu Asp Leu Thr Asp His Pro Val Thr 790 795 800 aga agt gaa aaggct gcc tcc ttt aaa ctg cag cgt cag aat cgt gtt 2565 Arg Ser Glu Lys AlaAla Ser Phe Lys Leu Gln Arg Gln Asn Arg Val 805 810 815 gac agc aaa gaaaca gag tgc taa tttagttctc agctcttctg acttaagtgt 2619 Asp Ser Lys GluThr Glu Cys 820 825 gcaaaatatt tttatagatt tgacctacaa tcaatcacagcttatatttt gtgaagactg 2679 ggaagtgact tagcagatgc tggtcatgtg tttgaacttcctgcaggtaa acagttcttg 2739 tgtggtttgg cccttctcct tttgaaaagg taaggtgaaggtgaatctag cttattttga 2799 ggctttcagg ttttagtttt taaaatatct tttgacctgtggtgcaaaag cagaaaatac 2859 agctggattg ggttatgagt atttacgttt ttgtaaattaatcttttata ttgataacag 2919 cactgactag ggaaatgatc agtttttttt ttatacactgtaatgaaccg ctgaatatga 2979 ggcatttggc atttatttgt gatgacaact ggaat 3014 220 DNA Artificial Sequence antisense sequence 2 cctagtcagt gctgttatca 203 20 DNA Artificial Sequence antisense sequence 3 gctgtgattg attgtaggtc20 4 20 DNA Artificial Sequence antisense sequence 4 agcatctgctaagtcacttc 20 5 20 DNA Artificial Sequence antisense sequence 5agctgagaac taaattagca 20 6 20 DNA Artificial Sequence antisense sequence6 tgagaactaa attagcactc 20 7 20 DNA Artificial Sequence antisensesequence 7 ttagcactct gtttctttgc 20 8 20 DNA Artificial Sequenceantisense sequence 8 ctgcagttta aaggaggcag 20 9 20 DNA ArtificialSequence antisense sequence 9 ctgtcaacac gattctgacg 20 10 20 DNAArtificial Sequence antisense sequence 10 aaatgacttg gcagctgtgc 20 11 20DNA Artificial Sequence antisense sequence 11 aaccacgctg gtcaggaata 2012 20 DNA Artificial Sequence antisense sequence 12 ataggagagactgcctcatg 20 13 20 DNA Artificial Sequence control sequence 13cctagtatgt gctgctatca 20 14 20 DNA Artificial Sequence control sequence14 ggatgttcgt cctcctcaca 20 15 24 DNA Artificial Sequence PCR primer 15gtgtcctact gcacaggtaa tagc 24 16 23 DNA Artificial Sequence PCR primer16 aatgacttgg cagctgtgct gct 23 17 11 PRT Artificial Sequence Peptidefragment 17 Arg Ala Asp Pro Asp Pro Met Lys Asn Thr Cys 1 5 10

What is claimed is:
 1. A method of inhibiting L-selectin shedding incells or tissue comprising contacting said cells or tissue in vitro withan antisense compound 8 to 30 nucleotides in length targeted to a 3′untranslated region, or a stop codon region of a nucleic acid moleculeencoding human TNF-α converting enzyme, wherein said antisense compoundinhibits human TNF-α converting enzyme expression or activity.
 2. Themethod of claim 1 wherein said antisense compound is an oligonucleotide.3. The method of claim 2, wherein said oligonucleotide is an enzymaticoligonucleotide targeted to a nucleic acid molecule encoding human TNF-αconverting enzyme, wherein said enzymatic oligonucleotide inhibits theexpression of human TNF-α converting enzyme.
 4. The method of claim 2,wherein said oligonucleotide comprises a modified internucleosidelinkage.
 5. The method of claim 4, wherein said modified internucleosidelinkage is a phosphorothioate linkage.
 6. The method of claim 2, whereinsaid oligonucleotide comprises at least one 2′-modified sugar moiety. 7.The method of claim 6, wherein said modification is 2′-O-methoxyethyl.8. The method of claim 2, wherein said oligonucleotide comprises atleast one modified base.
 9. The method of claim 8, wherein said modifiedbase is 5-methylcytosine.
 10. The method of claim 2 wherein saidoligonucleotide is a chimeric oligonucleotide.
 11. The method of claim 1wherein said antisense compound has a sequence comprising at least anβ-nucleobase portion of SEQ ID NO: 2, 3, 4, 5, 6, 7, or 12 and whereinsaid antisense compound inhibits human TNF-α converting enzymeexpression or activity.
 12. An antisense compound lip to 30 nucleobasesin length comprising at least an 8-nucleobase portion of SEQ ID NO: 8,9, 10 or 11 wherein said antisense compound inhibits TNF-α convertingenzyme expression or activity.
 13. The antisense compound of claim 12,wherein said antisense compound is an antisene oligonucleotide.
 14. Theantisense compouni of claim 13, wherein said oligonucleotide comprises amodified internucleoside linkage.
 15. The antisense compound of claim14, wherein said modified internucleoside linkage is a phosphorothioatelinkage.
 16. The antisense compound of claim 13, wherein saidoligonucleotide comprises at least one 2′-modified sugar moiety.
 17. Theantisenoe compound of claim 16, wherein said modification is2′-O-methoxyethyl.
 18. The antisense compound of claim 13, wherein saidoligonucleotide comprises at least one modified base.
 19. The antisensecompound of claim 18, wherein said modified base is 5-methylcytosine.20. The antisense compound of claim 13 wherein said oligonucleotide is achimeric oligonucleotide.